Dnase I digestion - (Aug/04/2005 )
Hi, I am new in this forum. It is a nice place to let people discuss the problem and share the invention.
I am recently working on the Dnase I digestion. However I have encount the problem with the digestion. I would like to know how much DNA I should be using ? and How many unit of Dnase I is good for the experiment? I have been reading a few papers and they all suggest different amount. I am confuse!!
Thanks for any suggestion.
I am performing hypersensitive analysis of promoter by using MNase and DNaseI. The conditions per se u have to standardize according to the need of experiment.
I perform DNase I digestion of chromatin with increasing concentration from 25 units to 150 units. 1 unit concentration represents amount of enzyme required to digest 1mg DNA.
At 150-200 units of chromatin I get 10 bp ladder.
Also i have observed that enzyme activity varies with different lot.
I have encounter the problem of standardize the chromatin. Do we use A260/280 to measure it ? or is there any other method? The reason is because once I lysis the nuclei, I have to do the digestion as soon as possible.
Beside, I would like to know if we use isolated DNA, would it affect the digestion result? since the conformation status might be changed after isolation.
I am not getting what experiment actually u are performing. Then only u can decide the starting material i.e. chromatin or DNA.
Actually i isolate nuclei and then estimate by A260 for DNA conc. of chromatin. I take 1ul of isolated nuclei in 99ul of 2MNaCl-5M urea ie 100X dilution (This lyses nuclei) and vortex thoroughly. and then perform DNA estimation. After this i perform DNase digestion of nuclei of known DNA conc.
Of coz digestion of naked DNA will be faster than that of chromatin.
Hope it might be of some help 2 u.